Transcription Factor HNF1 Research Articles

Inhibition of MMH (Met murine hepatocyte) cell differentiation by TGF(beta) is abrogated by pre-treatment with the heritable differentiation effector FGF1.

MMH (Met murine hepatocyte) liver cells derived from transgenic mice expressing a truncated constitutively active form of human c-Met are non-transformed immortalized cell lines. We have previously shown that they harbor: (1) epithelial cells that express the liver-enriched transcription factors HNF4 and HNF1(alpha), and that can be stably induced by FGF1 to express liver functions, and (2) fibroblast-like bi-potential palmate cells that can differentiate into bile duct-like structures in Matrigel cultures, or into epithelial cells competent to express hepatic functions. Low concentrations of TGF(beta) have been found to inhibit growth and differentiation of MMH cells. The factor stabilized the palmate cell phenotype, and it provoked epithelial cells to acquire palmate-like morphological characteristics, in parallel with down-regulation of expression of HNF4 and HNF1(alpha) and activation of Snail transcripts. The effects of TGF(beta) were dominant if it was added with FGF1, but the effects on differentiation were abrogated if cells had been pre-treated with FGF1. This work identifies TGF(beta) as a factor that could be implicated in maintaining bi-potential precursor cells in the liver, FGF1 as one that could over-ride the TGF(beta) effects and Snail as a candidate for mediation of the signal.

Identification of an intronic regulatory element in the human protein C (PROC) gene.

Regulatory DNA elements responsible for human protein C (PROC) gene expression have previously been identified in the upstream promoter region and first (untranslated) exon of the gene. Here we show that an additional sequence element located more than 500 bp downstream of the core promoter within intron 1 further enhances PROC promoter-driven reporter gene expression in human hepatoma cells. In common with core promoter constructs used in previous studies, the activity of this 3'-extended regulatory region is diminished by a naturally occurring promoter mutation. However, in contrast to constructs lacking intronic sequence, the promoter/intron regulatory region is repressed rather than activated by the transcription factor HNF-1. Using both conventional alignment procedures and complexity analysis to study the human and canine PROC sequences, we identified two conserved intronic regions, which were tested for their involvement in gene regulation. High-level gene expression from the intron-coupled promoter was dependent upon the integrity of a 142 bp sequence element, a duplicate copy of which is located in an upstream region of the PROC gene that possesses enhancer activity. These findings emphasise the potential importance of intragenic sequences for gene regulation and serve to illustrate that the results of PROC promoter/reporter gene experiments are critically dependent upon the sequence context. The identification of such intragenic elements is relevant to the analysis of human genetic disease since it will facilitate the detection and functional evaluation of regulatory mutations and polymorphisms.

Determinants of Vitellogenin B1 Promoter Architecture: HNF3 AND ESTROGEN RESPONSIVE TRANSCRIPTION WITHIN CHROMATIN

The liver-specific vitellogenin B1 promoter is efficiently activated by estrogen within a nucleosomal environment after microinjection into Xenopus laevis oocytes, consistent with the hypothesis that significant nucleosome remodeling over this promoter is not a prerequisite for the activation by the estrogen receptor (ERalpha). This observation lead us to investigate determinants other than ERalpha of chromatin structure and transcriptional activation of the vitellogenin B1 promoter in this system and in vitro. We find that the liver-enriched transcription factor HNF3 has an important organizational role for chromatin structure as demonstrated by DNase I-hypersensitive site mapping. Both HNF3 and the estrogen receptor activate transcription synergistically and are able to interact with chromatin reconstituted in vitro with three positioned nucleosomes. We propose that HNF3 is the cellular determinant which establishes a promoter environment favorable to a rapid transcriptional activation by the estrogen receptor.

A Winged Helix Protein from Yeast Saccharomyces cerevisiae Recognizes Centromere Sequences

The winged helix–turn–helix motif was initially identified in the mammalian hepatocyte-enriched transcription factor HNF-3 and the Drosophila forkhead homeotic protein. Proteins containing the winged helix motif have been shown to play important roles in tissue-specific developmental regulation. In this report, by using a genomic binding site selection method, we demonstrate that the winged helix protein YFKH-1 from the yeast Saccharomyces cerevisiae recognizes conserved sequence in yeast centromeres. Thus, our data suggest that the winged helix proteins of the yeast may be involved in centromeric functions of the yeast.

Repression and activation of transcription of phosphoenolpyruvate carboxykinase gene during liver development

Transcriptional activation of the hepatic phosphoenolpyruvate carboxykinase (PEPCK) gene at birth is critical since PEPCK appearance initiates hepatic gluconeogenesis. A delayed appearance results in hypoglycemia, while a premature appearance results in neonatal diabetes, both are incompatible with sustaining life. Experiments using transgenic mice and transfected hepatoma cells suggest that both repression and activation underlie the correct onset of hepatic PEPCK gene transcription. In transgenic mice, transgenes driven by the proximal PEPCK promoter are prematurely expressed in the fetal liver and over-expressed in the neonatal liver, indicating that sequences upstream of the proximal promoter restrain perinatal expression. In Hepa1c1c7 cells, which mimic the fetal liver, the proximal PEPCK promoter (597 bp) exhibited a 3.5–10-fold higher activity than longer promoters. Repression of the longer promoter (2000 bp) was diminished upon deletion of the sequence spanning positions −840 to −1116 which contains a PPAR/RXR recognition element. The intact 2000 bp PEPCK promoter could be markedly activated by co-transfecting the transcription factor HNF-1 together with C/EBP. It could be repressed by co-transfection with RXRα and adding PPARα relieved this inhibition.

Hepatocyte Nuclear Factor 1 Binds to and Transactivates the Human but Not the Rat CYP7A1 Promoter

Cholesterol 7α-hydroxylase (CYP7A1), a liver-specific enzyme, catalyzes the rate-limiting step in the degradation pathway of cholesterol to bile acids, and thus plays a key role in cholesterol homeostasis. To elucidate the mechanisms that control hepatic expression of the human CYP7A1 gene, we are studying the promoter region. Initially, we observed that up to 40% of the overall transcriptional activity of the promoter in HepG2 cells was associated with DNA sequences from −65 to −1 of the human gene. Within this region, a binding site for the liver-enriched transcription factor HNF-1 (−56 to −49) has been identified. Binding of HNF-1 to this site leads to transcriptional activation of the human promoter. The corresponding segment from the rat CYP7A1 gene does not bind HNF-1; instead, it is bound by the orphan receptors ARP-1 (COUP-TFII) and LXRα, that are implicated in dietary regulation.

Hepatocyte nuclear factor 3 (winged helix domain) activates trefoil factor gene TFF1 through a binding motif adjacent to the TATAA box.

The winged helix transcription factors HNF-3/FKH (forkhead homologs) activate endodermal-derived and acute-phase gene expression and control gut development in Drosophila. Trefoil factor family (TFFs) peptides are vertebrate products secreted by mucin-producing epithelial cells of the gastrointestinal tract involved in restitution and repair of the mucosa. They are positively regulated in ulcerative and neoplastic conditions. We describe a consensus sequence in human and rodent TFF promoters close to the TATAA box showing striking similarity to the binding site of the HNF-3/FKH family. In gel retardation assays, HNF-3 alpha and beta bound predominantly to the site in TFF1 (formerly pS2) and, to a lesser extent, to the sites in TFF2 or TFF3. Mutations generated in this motif severely impaired transcription of TFF1 reporter genes. Cotransfection with expression vectors of HNF-3alpha and beta, but not the related HFH 11A and B, specifically activated the wild-type TFF1 reporter genes. Activation of endogenous expression of TFF1 by HNF-3 alpha and beta gene products was more than 1000 fold in the pancreatic cell line Capan-2 and fivefold in the gastric cell line MKN-45, whereas the intestinal cell lines HUTU 80 and HT-29 displayed no effect. Thus, HNF-3/FKH factors contribute causally to cell-specific regulation of TFF genes and may explain the acute-phase response of TFF peptides.

Exon-I is involved in positive as well as negative regulation of human angiotensinogen gene expression.

Angiotensinogen is the glycoprotein precursor of one of the most potent vasoactive hormones, angiotensin-II. Angiotensinogen gene is primarily expressed in the liver, and this gene locus is linked with human essential hypertension. We show here that a mutation in exon-I reduces the basal expression of the human angiotensinogen gene in liver cells. We also show that a nucleotide sequence in exon-I binds to liver-enriched transcription factor HNF-3 and a ubiquitous factor AP4. Our studies also show that transient transfection of an expression vector containing AP4 coding sequence downregulates the expression of reporter constructs containing human angiotensinogen gene promoter. By contrast, co-transfection of an expression vector containing HNF-3beta coding sequence increases the expression of these reporter constructs. The human angiotensinogen gene has a C/A polymorphism located at -20, and we have shown that estrogen receptor-alpha binds to this sequence when nucleoside A is present at this site. We show here that co-transfection of an expression vector containing AP4 coding sequence reduces estrogen-induced promoter activity of reporter constructs containing human angiotensinogen gene promoter (with nucleoside A at -20) attached to the CAT gene. These studies partly explain the molecular mechanisms involved in tissue-specific expression of the human angiotensinogen gene.

Identification of a bipotential precursor cell in hepatic cell lines derived from transgenic mice expressing cyto-Met in the liver.

Met murine hepatocyte (MMH) lines were established from livers of transgenic mice expressing constitutively active human Met. These lines harbor two cell types: epithelial cells resembling the parental populations and flattened cells with multiple projections and a dispersed growth habit that are designated palmate. Epithelial cells express the liver-enriched transcription factors HNF4 and HNF1alpha, and proteins associated with epithelial cell differentiation. Treatments that modulate their differentiation state, including acidic FGF, induce hepatic functions. Palmate cells show none of these properties. However, they can differentiate along the hepatic cell lineage, giving rise to: (a) epithelial cells that express hepatic transcription factors and are competent to express hepatic functions; (b) bile duct-like structures in three-dimensional Matrigel cultures. Derivation of epithelial from palmate cells is confirmed by characterization of the progeny of individually fished cells. Furthermore, karyotype analysis confirms the direction of the phenotypic transition: palmate cells are diploid and the epithelial cells are hypotetraploid. The clonal isolation of the palmate cell, an immortalized nontransformed bipotential cell that does not yet express the liver-enriched transcription factors and is a precursor of the epithelial-hepatocyte in MMH lines, provides a new tool for the study of mechanisms controlling liver development.

Growth and Differentiation of Rat Hepatocytes: Changes in Transcription Factors HNF-3, HNF-4, STAT-3, and STAT-5

The liver enriched transcription factors HNF-3 and HNF-4 are known to play major roles in development and differentiation of hepatocytes. STAT-3 and STAT-5 are signaling peptides activated by a variety of cytokines and growth factors including HGF and EGF. Their role in hepatocyte growth and differentiation is yet to be determined. We examined protein expression and DNA binding activities of these transcription factors in a hepatocyte culture system in which the hepatocytes first de-differentiate and proliferate. Overlaying proliferating hepatocytes with EHS-matrix led to an increase in HNF-4 protein and DNA-binding activity. STAT-5 DNA binding activity was only slightly effected by EHS-matrix. HNF-3 and STAT-3 DNA-binding activities were reduced in the presence of EHS-matrix. This is consistent with the role of HNF-3 as the major initiating transcription factor involved in embryonic liver development and suggests, that STAT-3 might also play a role in growth and differentiation of hepatocytes.

Activated DNA- And RNA-Duplexes Covalently Cross-Linked to Regulatory Proteins

A novel class of DNA- and RNA duplexes including trisubstituted pyrophosphate internucleotide groups (TSP) in sugar-phosphate backbone was employed in cross-linking to proteins. Substrates including TSP effectively bound covalently to nucleofilic aminoacids (Lys, His) of transcription factors HNF1 and NF-kB and to HIV-1 Tat protein without additional activation at physiological conditions. FAX: (095)939–3181: E-mail: zoeshab@nuclacid.genebee.msu.su

Phenobarbital Suppresses Growth and Accelerates Restoration of Differentiation Markers of Primary Culture Rat Hepatocytes in the Chemically Defined Hepatocyte Growth Medium Containing Hepatocyte Growth Factor and Epidermal Growth Factor

Phenobarbital (PB), a liver-tumor promoter, at a concentration of 3 mM dramatically inhibited the growth of adult rat hepatocytes in the chemically defined medium, HGM, with added hepatocyte growth factor (HGF) and epidermal growth factor (EGF). In concurrence with these findings, PB down-regulated expression of the HGF receptor (c-met) and suppressed production of the autocrine growth factor transforming growth factor-α (TGF-α). Furthermore, PB down-regulated expression of transcription factors associated with proliferation such as AP1 and NF-κB. In the presence of PB, hepatocytes remained morphologically differentiated and restoration of the expression of mature hepatocyte markers, such as albumin and cytochrome P450s (1A, 2B1/2, and 2E1), was accelerated after an initial phase of growth. Additionally, PB strongly suppressed expression of the mRNA for α-fetoprotein, a protein primarily expressed by fetal liver, and the accelerative effect of PB on restoration of mature hepatocyte markers showed a correlation with the up-regulation of the hepatocyte-enriched transcription factors HNF3 and HNF4. When the effects of PB on various extracellular matrix proteins were examined, the data indicated that PB specifically suppressed laminin and fibronectin production by hepatocytes, suggesting an important role for these proteins in growing hepatocyte cultures.

Binding of the winged-helix transcription factor HNF3 to a linker histone site on the nucleosome.

The transcription factor HNF3 and linker histones H1 and H5 possess winged-helix DNA-binding domains, yet HNF3 and other fork head-related proteins activate genes during development whereas linker histones compact DNA in chromatin and repress gene expression. We compared how the two classes of factors interact with chromatin templates and found that HNF3 binds DNA at the side of nucleosome cores, similarly to what has been reported for linker histone. A nucleosome structural binding site for HNF3 is occupied at the albumin transcriptional enhancer in active and potentially active chromatin, but not in inactive chromatin in vivo. While wild-type HNF3 protein does not compact DNA extending from the nucleosome, as does linker histone, site-directed mutants of HNF3 can compact nucleosomal DNA if they contain basic amino acids at positions previously shown to be essential for nucleosomal DNA compaction by linker histones. The results illustrate how transcription factors can possess special nucleosome-binding activities that are not predicted from studies of factor interactions with free DNA.

Nucleosome positioning by the winged helix transcription factor HNF3.

Nucleosome positioning at genetic regulatory sequences is not well understood. The transcriptional enhancer of the mouse serum albumin gene is active in liver, where regulatory factors occupy their target sites on three nucleosome-like particles designated N1, N2, and N3. The winged helix transcription factor HNF3 binds to two sites near the center of the N1 particle. We created dinucleosome templates using the albumin enhancer sequence and found that site-specific binding of HNF3 protein resulted in nucleosome positioning in vitro similar to that seen in liver nuclei. Thus, binding of a transcription factor can position an underlying nucleosome core.

A set of polyclonal and monoclonal antibodies reveals major differences in post-translational modification of the rat HNF1 and vHNF1 homeoproteins

The related homeodomain-containing transcription factors HNF1 (HNF1α) and vHNF1 (HNF1β) recognise common target DNA sequences in the regulatory regions of many genes and are expressed in several parenchymal cell types, predominantly in liver, kidney, intestine and pancreas. HNFI-mull mutant mice, with a wild-type vHNF1 gene, develop normally, but die within a few weeks of birth with severe liver and kidney failure. Humans with a mutation in the HNF1α gene develop non-insulin dependent diabetes on maturity (MODY 3). To determine distinctive roles for each of these proteins we produced a set of polyclonal sera and monoclonal antibodies, directed against different parts of the rat HNF1 and vHNF1 proteins. These antibodies reveal that HNF1 is present in vivo as a heterogeneous mixture of 92–98 kDa molecular mass polypeptides, a mass higher than that expected from its amino acid sequence, vHNF1 is present in the form of two isoforms of roughly the expected molecular masses, 65 and 68 kDa. In addition, some antibodies prepared against bacterially produced HNF1 recognise vHNF1 but not HNF1, in liver and kidney extracts. Hence, we present the first evidence for differential post-translational modification of HNF1 and vHNF1 proteins.

Cerberus-like is a secreted factor with neuralizing activity expressed in the anterior primitive endoderm of the mouse gastrula

We report the isolation of mouse cerberus-like ( cer-l), a gene encoding a novel secreted protein that is specifically expressed in the anterior visceral endoderm during early gastrulation. Expression in the primitive endoderm starts before the appearance of the primitive streak and lasts until the head-fold stage. In later stages, a second region of expression is found in newly formed somites. Mouse cer-l shares some sequence similarity with Xenopus cerberus ( Xcer). In Xenopus assays cer-l, like Xcer, mRNA acts as a potent neuralizing factor that induces forebrain markers and endoderm, but is unable to induce ectopic head-like structures as Xcer does. In addition to cer-l, anterior visceral endoderm was found to express the transcription factors Lim1, goosecoid and HNF-3 β that are also present in trunk organizer cells. A model of how head and trunk development might be regulated is discussed. Given its neuralizing activity, the secreted protein Cer-l is a candidate for mediating inductive activities of anterior visceral endoderm.

The apolipoprotein A-I/C-III/A-IV gene cluster: ApoC-III and ApoA-IV expression is regulated by two common enhancers

Genetic, epidemiological and clinical evidence have clearly demonstrated the importance of the human apolipoprotein (apo) A-I/C-III/A-IV gene cluster in lipid metabolism and heart attack. The transcriptional regulation of these genes determines the level of the encoded proteins and therefore influences the concentration of triglycerides and cholesterol. Here, we analyze the existence of transcription control elements in the 6.6 kb apoC-III/A-IV intergenic region and their influence on the expression of both genes. Two main positive common control elements were found to modulate apoC-III and apoA-IV expression in HepG2 and in Caco-2 cells: the previously described apoC-III enhancer, located 0.8 kb upstream from the cap site of the gene, and a newly detected activating region located in the center of the intergenic sequence. The activity of both elements is highly increased by the hepatic and intestinal transcription factor HNF-4. Analysis of a 641 bp fragment containing the central element showed that it has the properties of a tissue-specific enhancer. Liver nuclear proteins interact with seven DNA binding sites present in this enhancer and HNF-4 specifically interacts with one of these sites. A third positive element, situated immediately upstream from the apoA-IV minimal promoter, is also activated by HNF-4; however, this element is not involved in apoC-III expression. In addition, two negative regions were identified, one located near the apoA-IV gene and the other one between the apoC-III enhancer and the newly identified central enhancer. In conclusion, negative and positive control elements are located in the apoC-III/A-IV intergenic region, including two enhancers important for the expression of the two genes. These results add new evidence that common regulatory elements for the expression of the apoA-I, apoC-III and apoA-IV genes are interspersed throughout the cluster.

Activation of CAAT Enhancer-binding Protein δ (C/EBPδ) by Interleukin-1 Negatively Influences Apolipoprotein C-III Expression

Tissue-specific transcription of the apolipoprotein C-III (apoC-III) gene is mainly regulated by synergistic interactions between the liver-enriched transcription factor HNF-4, which binds to the proximal promoter, and ubiquitous factors, which bind to the upstream enhancer region. Here we show that apoC-III expression in HepG2 cells is negatively regulated in response to interleukin-1 (IL-1), and this inhibition is mainly due to transcriptional repression. CAAT enhancer-binding protein delta (C/EBPdelta) was found to be the main mediator of IL-1-induced suppression. Analysis of the apoC-III promoter revealed two IL-1 response elements. The first is located in the proximal promoter region D and the second in the distal enhancer region I. Proximal element D is a high affinity binding site for C/EBPdelta, while the enhancer element I is not directly recognized by this transcription factor. Functional analysis of different combinations of homologous and heterologous promoter constructs revealed that indirect interaction of C/EBPdelta with site I, in the context of the full promoter, leads to repression. C/EBPdelta is activated by phosphorylation during IL-1-induced signal transduction pathway. This modification is important for both DNA binding activity and indirect transrepression of the apoC-III promoter.

Activity and Inducibility of Drug-Metabolizing Enzymes in Immortalized Hepatocyte-like Cells (mhPKT) Derived from al-PK/Tag1 Transgenic Mouse

This report describes the establishment and characterization of the mhPKT cell line derived from the liver of a transgenic mouse harboring the simian virus (SV40) large T and small t antigens placed under the control of the 5′ regulatory sequence of the ratl-type pyruvate kinase (l-PK) gene. mhPKT cells had a prolonged life span, expressed the SV40-encoded nuclear large T antigen when grown in glucose-enriched medium, and induced tumors when injected subcutaneously into athymic (nu-nu) mice. Growth on petri dishes or filters yielded multiple layers of cuboid cells, with numerous spaces between adjacent cells that were closed by junctional complexes. These bile canaliculi-like structures exhibited numerous microvilli in which villin, an actin-binding brush-border protein, colocalized with actin. These bile canaliculi-like structures appeared to be functional as they accumulated fluorescein. mhPKT cells conserved the expression of the liver-specific transcription factors HNF1, HNF3, HNF4, and DBP together with substantial levels ofl-PK and albumin but not α-fetoprotein mRNA transcripts. mhPKT cells mainly metabolized testosterone into androstenedione and 6β-hydroxytestosterone, asin vivo.3-Methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) markedly increased ethoxyresorufin-O-deethylase activity and the related cytochrome P450 (CYP) 1A1/2 protein, whereas α-naphtoflavone antagonized the TCDD-elicited induction. Phenobarbital slightly increased the CYP2B-mediated activities of pentoxyresorufin-O-depentylase, 2β- and 16β-testosterone hydroxylase. mhPKT cells also had substantial sulfotransferase, UDP-glucuronyltransferase, and glutathioneS-transferase activities. This model may serve as a tool for long-termin vitrostudies of xenobiotic metabolism, potent CYP inducers, and hepatocyte damage due to drugs and other factors.

Transcriptional regulation by HNF-4 of the steroid 15α-hydroxylase P450 ( Cyp2a-4) gene in mouse liver

The mouse P450 gene Cyp2a-4 encodes the hepatic steroid 15α-hydroxylase. We have defined in the 5′-flanking sequence of Cyp2a-4 gene, a composite regulatory element ( −61AGACCAAAGTC CG GCCTTC −42) which contains a potential CpG methylation site at position −50. Gel-shift assays indicate that this element consists of overlapped binding sites for a hepatocyte-enriched transcription factor HNF-4 and a Sp1-like protein. Moreover, transcription of the Cyp2a-4 gene is activated by coexpression of HNF-4 in HepG2 cells. A mutation (C at −50 to A) abolishes the binding of HNF-4 to the element as well as the transcriptional activation by HNF-4. The methylated C at position −50, however, does not affect HNF-4 binding. Neither the mutation nor the methylation at position −50 affect the binding of Sp1-like protein to the element. It appears, therefore, that HNF-4 activates the hepatic transcription of Cyp2a-4 gene through its direct binding to the regulatory element regardless of the methylation at position −50.